A 'classical' molecular genetic approach to mammalian gene expression involves the analysis of naturally-occurring variations among closely related strains or species. This approach is a powerful one from two perspectives. First, it identifies novel genomic alterations that modulate gene expression, thereby providing unique information on the molecular mechanisms governing gene activity. Second, in being essentially an evolutionary approach, it defines critical molecular targets for evolutionary change. Our laboratory has been studying renal gene expression in the mouse (genus Mus). The transcription rates of a number of genes are induced by androgens in the mouse kidney; importantly, extensive variations in the inducibility of these genes occur among Mus species, signalling the existence of gene- and species-- specific regulatory mutations. One such gene, called RP2, is differentially responsive to androgens in M. domesticus and M. caroli; based upon recent experimental results, we have proposed a molecular model to explain the inter-species difference. In the current application, we will focus on verifying and extending this model, using a variety of experimental strategies. We will examine DNA sequences near the RP2 gene and will define the nuclear proteins that bind them; M. domesticus and M. caroli will be compared in these regards. The DNA binding factors that we have proposed as responsible for the inter-species difference will be purified and characterized, their corresponding genes will be cloned, and their regulation will be analyzed at the level of mRNA. Several experimental systems for testing the function of regulatory elements that may be important to RP2 expression and its species-specific regulation in kidney will be developed. Finally, we will examine additional species within the Mus genus to gain insights into the nature and extent of evolutionary modification of molecular mechanisms for RP2 gene control. We believe that further studies of renal gene expression in mice will generate fundamental new information relating to the identity and action of molecular factors that control gene activity and that drive the evolution of species-specific regulatory mechanisms.